Detection of group B Streptococcus

ABSTRACT

The invention provides methods to detect group B  streptococcus  (GBS) in biological samples using real-time PCR. Primers and probes for the detection of GBS are provided by the invention. Articles of manufacture containing such primers and probes for detecting GBS are further provided by the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §120 of U.S.application Ser. No. 10/716,005 filed on Nov. 18, 2003, which issued asU.S. Pat. No. 7,427,475 on Sep. 23, 2008.

TECHNICAL FIELD

This invention relates to bacterial diagnostics, and more particularlyto detection of group B streptococcus (GBS).

BACKGROUND

Group B Streptococcus (GBS) is a Gram positive bacteria commonly foundin the throat and lower intestine of adults, and in the vagina of women.Normally, this organism does not cause disease in the host. Duringchildbirth, however, an infant can become infected with GBS. GBSinfections are the leading cause of neonatal morbidity and mortality inthe United States, with fatality ratios as high as 50% in untreatedcases. In recent years, antibiotics administered during labor havegreatly reduced the incidence of neonatal GBS.

Current CDC recommendations call for screening of women for GBS duringweek 35 to 37 weeks' gestation by culture. Women found to be colonizedwith GBS are treated with intravenous antibiotics during labor. Thisapproach has reduced the incidence of neonatal infection and fewerpatients are provided unnecessary antibiotic treatment. However, theproblem of GBS neonatal sepsis has not been eliminated. Colonizationwith GBS is often transient, so lack of colonization at 35 weeksgestation does not guarantee that there will be no GBS present at week40. Also, many patients present to healthcare providers first at thetime of labor and have not been screened for GBS. The decision to treatwith antibiotics in these cases must be made on the basis of riskfactors such as gestation <37 weeks, membrane rupture >12 hours, youngmaternal age, and/or black or Hispanic ethnicity.

Ideally, the determination of GBS colonization would be made duringearly labor and the laboratory results available within a few hours.Conventional identification of GBS requires culture. As culture mayrequire up to 72 hours for a definitive answer, physicians may provideunnecessary antimicrobics at the time of delivery on an empiric basis.Overuse of antimicrobics may predispose to the development ofantimicrobial resistance and add to the risk of adverse reactionsincluding life-threatening anaphylaxis. Rapid antigen tests (e.g., latexagglutination) also have been used to diagnose GBS, but these tests lacksensitivity when compared to culture. In fact, the sensitivities ofantigen tests for detecting GBS are so low that the FDA has issued analert that these types of assays cannot be used to diagnose GBS withoutculture backup.

SUMMARY

The invention provides for methods of identifying group B streptococcus(GBS) in a biological sample or in a non-biological sample. Primers andprobes for detecting GBS are provided by the invention, as are kitscontaining such primers and probes. Methods of the invention can be usedto rapidly identify GBS DNA from specimens for diagnosis of GBSinfection. Using specific primers and probes, the methods includeamplifying and monitoring the development of specific amplificationproducts using fluorescence resonance energy transfer (FRET).

In one aspect of the invention, there is provided a method for detectingthe presence or absence of GBS in a biological sample from an individualor in a non-biological sample. The method to detect GBS includesperforming at least one cycling step, which includes an amplifying stepand a hybridizing step. The amplifying step includes contacting thesample with a pair of pts primers to produce a pts amplification productif a GBS pts nucleic acid molecule is present in the sample. Thehybridizing step includes contacting the sample with a pair of ptsprobes. Generally, the members of the pair of pts probes hybridizewithin no more than five nucleotides of each other. A first pts probe ofthe pair of pts probes is typically labeled with a donor fluorescentmoiety and a second pts probe of the pair of pts probes is labeled witha corresponding acceptor fluorescent moiety. The method further includesdetecting the presence or absence of FRET between the donor fluorescentmoiety of the first pts probe and the acceptor fluorescent moiety of thesecond pts probe. The presence of FRET is usually indicative of thepresence of GBS in the sample, while the absence of FRET is usuallyindicative of the absence of GBS in the sample.

A pair of pts primers generally includes a first pts primer and a secondpts primer. The first pts primer can include the sequence 5′-TGA GAA GGCAGT AGA AAG CTT AG-3′ (SEQ ID NO:1), and the second pts primer caninclude the sequence 5′-TGC ATG TAT GGG TTA TCT TCC-3′ (SEQ ID NO:2). Afirst pts probe can include the sequence 5′-CAA ATT AAA GAG ACT ATT CGTGCA A-3′ (SEQ ID NO:3), and the second pts probe can include thesequence 5′-CAA GTA AAT GCA GAA ACA GG-3′ (SEQ ID NO:4).

In some aspects, one of the pts primers can be labeled with afluorescent moiety (either a donor or acceptor, as appropriate) and cantake the place of one of the pts probes.

The members of the pair of pts probes can hybridize within no more thantwo nucleotides of each other, or can hybridize within no more than onenucleotide of each other. A representative donor fluorescent moiety isfluorescein, and corresponding acceptor fluorescent moieties includeLC-Red 640, LC-Red 705, Cy5, and Cy5.5. Additional corresponding donorand acceptor fluorescent moieties are known in the art.

In one aspect, the detecting step includes exciting the sample at awavelength absorbed by the donor fluorescent moiety and visualizingand/or measuring the wavelength emitted by the acceptor fluorescentmoiety (i.e., visualizing and/or measuring FRET). In another aspect, thedetecting step includes quantitating the FRET. In yet another aspect,the detecting step can be performed after each cycling step (e.g., inreal-time).

Generally, the presence of FRET within 45 cycles (e.g., 20, 25, 30, 35,or 40 cycles) indicates the presence of a GBS infection in theindividual. In addition, determining the melting temperature between oneor both of the pts probe(s) and the pts amplification product canconfirm the presence or absence of the GBS.

Representative biological sample include anal and/or vaginal swabs. Theabove-described methods can further include preventing amplification ofa contaminant nucleic acid. Preventing amplification can includeperforming the amplifying step in the presence of uracil and treatingthe sample with uracil-DNA glycosylase prior to amplifying.

In addition, the cycling step can be performed on a control sample. Acontrol sample can include the same portion of the GBS pts nucleic acidmolecule. Alternatively, a control sample can include a nucleic acidmolecule other than a GBS pts nucleic acid molecule. Cycling steps canbe performed on such a control sample using a pair of control primersand a pair of control probes. The control primers and probes are otherthan pts primers and probes. One or more amplifying steps produces acontrol amplification product. Each of the control probes hybridizes tothe control amplification product.

In another aspect of the invention, there are provided articles ofmanufacture, or kits. Kits of the invention can include a pair of ptsprimers, and a pair of pts probes, and a donor and correspondingacceptor fluorescent moieties. For example, the first pts primerprovided in a kit of the invention can have the sequence 5′-TGA GAA GGCAGT AGA AAG CTT AG-3′ (SEQ ID NO:1) and the second pts primer can havethe sequence 5′-TGC ATG TAT GGG TTA TCT TCC-3′ (SEQ ID NO:2). The firstpts probe provided in a kit of the invention can have the sequence5′-CAA ATT AAA GAG ACT ATT CGT GCA A-3′ (SEQ ID NO:3) and the second ptsprobe can have the sequence 5′-CAA GTA AAT GCA GAA ACA GG-3′ (SEQ IDNO:4).

Articles of manufacture can include fluorophoric moieties for labelingthe probes or probes already labeled with donor and correspondingacceptor fluorescent moieties. The article of manufacture can alsoinclude a package insert having instructions thereon for using theprimers, probes, and fluorophoric moieties to detect the presence orabsence of GBS in a sample.

In yet another aspect of the invention, there is provided a method fordetecting the presence or absence of GBS in a biological sample from anindividual or in a non-biological sample. Such a method includesperforming at least one cycling step. A cycling step can include anamplifying step and a hybridizing step. Generally, an amplifying stepincludes contacting the sample with a pair of pts primers to produce apts amplification product if a GBS pts nucleic acid molecule is presentin the sample. Generally, a hybridizing step includes contacting thesample with a pts probe. Such a pts probe is usually labeled with adonor fluorescent moiety and a corresponding acceptor fluorescentmoiety. The method further includes detecting the presence or absence offluorescence resonance energy transfer (FRET) between the donorfluorescent moiety and the acceptor fluorescent moiety of the pts probe.The presence or absence of fluorescence is indicative of the presence orabsence of GBS in said sample.

In one aspect, amplification can employ a polymerase enzyme having 5′ to3′ exonuclease activity. Thus, the first and second fluorescent moietieswould be within no more than 5 nucleotides of each other along thelength of the probe. In another aspect, the pts probe includes a nucleicacid sequence that permits secondary structure formation. Such secondarystructure formation generally results in spatial proximity between thefirst and second fluorescent moiety. According to this method, thesecond fluorescent moiety on a probe can be a quencher.

In another aspect of the invention, there is provided a method fordetecting the presence or absence of GBS in a biological sample from anindividual or in a non-biological sample. Such a method includesperforming at least one cycling step. A cycling step can include anamplifying step and a dye-binding step. An amplifying step generallyincludes contacting the sample with a pair of pts primers to produce apts amplification product if a GBS pts nucleic acid molecule is presentin the sample. A dye-binding step generally includes contacting the ptsamplification product with a double-stranded DNA binding dye. The methodfurther includes detecting the presence or absence of binding of thedouble-stranded DNA binding dye into the amplification product.According to the invention, the presence of binding is typicallyindicative of the presence of GBS in the sample, and the absence ofbinding is typically indicative of the absence of GBS in the sample.Such a method can further include the steps of determining the meltingtemperature between the pts amplification product and thedouble-stranded DNA binding dye. Generally, the melting temperatureconfirms the presence or absence of GBS. Representative double-strandedDNA binding dyes include SYBRGreenI®, SYBRGold®, and ethidium bromide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedrawings and detailed description, and from the claims.

DETAILED DESCRIPTION

A real-time assay for detecting GBS in a biological sample or in anon-biological sample that is more sensitive and specific than existingassays is described herein. Primers and probes for detecting GBSinfections and articles of manufacture containing such primers andprobes are provided by the invention. The increased sensitivity ofreal-time PCR for detection of GBS compared to other methods, as well asthe improved features of real-time PCR including sample containment andreal-time detection of the amplified product, make feasible theimplementation of this technology for routine diagnosis of GBSinfections in the clinical laboratory.

GBS Nucleic Acids and Oligonucleotides

The invention provides methods to detect GBS by amplifying, for example,a portion of the GBS pts nucleic acid. GBS nucleic acids other thanthose exemplified herein (e.g., other than pts) also can be used todetect GBS in a sample and are known to those of skill in the art.Nucleic acid sequences from GBS are available (see, for example, GenBankAccession Nos. NC_(—)004368 and NC_(—)004116). Specifically, primers andprobes to amplify and detect GBS pts nucleic acid molecules are providedby the invention.

Primers that amplify a GBS nucleic acid molecule, e.g., GBS pts can bedesigned using, for example, a computer program such as OLIGO (MolecularBiology Insights, Inc., Cascade, Colo.). Important features whendesigning oligonucleotides to be used as amplification primers include,but are not limited to, an appropriate size amplification product tofacilitate detection (e.g., by electrophoresis), similar meltingtemperatures for the members of a pair of primers, and the length ofeach primer (i.e., the primers need to be long enough to anneal withsequence-specificity and to initiate synthesis but not so long thatfidelity is reduced during oligonucleotide synthesis). Typically,oligonucleotide primers are 15 to 30 nucleotides in length.

Designing oligonucleotides to be used as hybridization probes can beperformed in a manner similar to the design of primers, although themembers of a pair of probes preferably anneal to an amplificationproduct within no more than 5 nucleotides of each other on the samestrand such that FRET can occur (e.g., within no more than 1, 2, 3, or 4nucleotides of each other). This minimal degree of separation typicallybrings the respective fluorescent moieties into sufficient proximitysuch that FRET occurs. It is to be understood, however, that otherseparation distances (e.g., 6 or more nucleotides) are possible providedthe fluorescent moieties are appropriately positioned relative to eachother (for example, with a linker arm) such that FRET can occur. Inaddition, probes can be designed to hybridize to targets that contain apolymorphism or mutation, thereby allowing differential detection of GBSstrains based on either absolute hybridization of different pairs ofprobes corresponding to the particular GBS strain to be distinguished ordifferential melting temperatures between, for example, members of apair of probes and each amplification product corresponding to a GBSstrain to be distinguished. As with oligonucleotide primers,oligonucleotide probes usually have similar melting temperatures, andthe length of each probe must be sufficient for sequence-specifichybridization to occur but not so long that fidelity is reduced duringsynthesis. Oligonucleotide probes are generally 15 to 30 nucleotides inlength.

Constructs of the invention include vectors containing a GBS nucleicacid molecule, e.g., GBS pts or a fragment thereof. Constructs of theinvention can be used, for example, as control template nucleic acidmolecules. Vectors suitable for use in the present invention arecommercially available and/or produced by recombinant DNA technologymethods routine in the art. GBS pts nucleic acid molecules can beobtained, for example, by chemical synthesis, direct cloning from GBS,or by PCR amplification. A GBS nucleic acid molecule or fragment thereofcan be operably linked to a promoter or other regulatory element such asan enhancer sequence, a response element, or an inducible element thatmodulates expression of the GBS nucleic acid molecule. As used herein,operably linking refers to connecting a promoter and/or other regulatoryelements to a GBS nucleic acid molecule in such a way as to permitand/or regulate expression of the GBS nucleic acid molecule. A promoterthat does not normally direct expression of GBS pts can be used todirect transcription of a pts nucleic acid using, for example, a viralpolymerase, a bacterial polymerase, or a eukaryotic RNA polymerase II.Alternatively, the pts native promoter can be used to directtranscription of a pts nucleic acid. In addition, operably linked canrefer to an appropriate connection between a GBS pts promoter orregulatory element and a heterologous coding sequence (i.e., a non-ptscoding sequence, for example, a reporter gene) in such a way as topermit expression of the heterologous coding sequence.

Constructs suitable for use in the methods of the invention typicallyinclude, in addition to GBS pts nucleic acid molecules, sequencesencoding a selectable marker (e.g., an antibiotic resistance gene) forselecting desired constructs and/or transformants, and an origin ofreplication. The choice of vector systems usually depends upon severalfactors, including, but not limited to, the choice of host cells,replication efficiency, selectability, inducibility, and the ease ofrecovery.

Constructs of the invention containing GBS pts nucleic acid moleculescan be propagated in a host cell. As used herein, the term host cell ismeant to include prokaryotes and eukaryotes such as yeast, plant andanimal cells. Prokaryotic hosts may include E. coli, Salmonellatyphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hostsinclude yeasts such as S. cerevisiae, S. pombe, Pichia pastoris,mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells,insect cells, and plant cells such as Arabidopsis thaliana and Nicotianatabacum. A construct of the invention can be introduced into a host cellusing any of the techniques commonly known to those of ordinary skill inthe art. For example, calcium phosphate precipitation, electroporation,heat shock, lipofection, microinjection, and viral-mediated nucleic acidtransfer are common methods for introducing nucleic acids into hostcells. In addition, naked DNA can be delivered directly to cells (see,e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466).

Polymerase Chain Reaction (PCR)

U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 discloseconventional PCR techniques. PCR typically employs two oligonucleotideprimers that bind to a selected nucleic acid template (e.g., DNA orRNA). Primers useful in the present invention include oligonucleotidescapable of acting as a point of initiation of nucleic acid synthesiswithin GBS pts nucleic acid sequences. A primer can be purified from arestriction digest by conventional methods, or it can be producedsynthetically. The primer is preferably single-stranded for maximumefficiency in amplification, but the primer can be double-stranded.Double-stranded primers are first denatured, i.e., treated to separatethe strands. One method of denaturing double stranded nucleic acids isby heating.

The term “thermostable polymerase” refers to a polymerase enzyme that isheat stable, i.e., the enzyme catalyzes the formation of primerextension products complementary to a template and does not irreversiblydenature when subjected to the elevated temperatures for the timenecessary to effect denaturation of double-stranded template nucleicacids. Generally, the synthesis is initiated at the 3′ end of eachprimer and proceeds in the 5′ to 3′ direction along the template strand.Thermostable polymerases have been isolated from Thermus flavus, T.ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillusstearothermophilus, and Methanothermus fervidus. Nonetheless,polymerases that are not thermostable also can be employed in PCR assaysprovided the enzyme is replenished.

If the GBS template nucleic acid is double-stranded, it is necessary toseparate the two strands before it can be used as a template in PCR.Strand separation can be accomplished by any suitable denaturing methodincluding physical, chemical or enzymatic means. One method ofseparating the nucleic acid strands involves heating the nucleic aciduntil it is predominately denatured (e.g., greater than 50%, 60%, 70%,80%, 90% or 95% denatured). The heating conditions necessary fordenaturing template nucleic acid will depend, e.g., on the buffer saltconcentration and the length and nucleotide composition of the nucleicacids being denatured, but typically range from about 90° C. to about105° C. for a time depending on features of the reaction such astemperature and the nucleic acid length. Denaturation is typicallyperformed for about 30 sec to 4 min.

If the double-stranded nucleic acid is denatured by heat, the reactionmixture is allowed to cool to a temperature that promotes annealing ofeach primer to its target sequence on the GBS nucleic acid. Thetemperature for annealing is usually from about 35° C. to about 65° C.Annealing times can be from about 10 secs to about 1 min. The reactionmixture is then adjusted to a temperature at which the activity of thepolymerase is promoted or optimized, i.e., a temperature sufficient forextension to occur from the annealed primer to generate productscomplementary to the template nucleic acid. The temperature should besufficient to synthesize an extension product from each primer that isannealed to a nucleic acid template, but should not be so high as todenature an extension product from its complementary template (e.g., thetemperature for extension generally ranges from about 40° to 80° C.).Extension times can be from about 10 secs to about 5 mins.

PCR assays can employ GBS nucleic acid such as DNA or RNA, includingmessenger RNA (mRNA). The template nucleic acid need not be purified; itmay be a minor fraction of a complex mixture, such as GBS nucleic acidcontained in human cells. DNA or RNA may be extracted from a biologicalsample such as an anal and/or vaginal swab by routine techniques such asthose described in Diagnostic Molecular Microbiology: Principles andApplications (Persing et al. (eds), 1993, American Society forMicrobiology, Washington D.C.). Nucleic acids can be obtained from anynumber of sources, such as plasmids, or natural sources includingbacteria, yeast, viruses, organelles, or higher organisms such as plantsor animals.

The oligonucleotide primers are combined with PCR reagents underreaction conditions that induce primer extension. For example, chainextension reactions generally include 50 mM KCl, 10 mM Tris-HCl (pH8.3), 15 mM MgCl₂, 0.001% (w/v) gelatin, 0.5-1.0 μg denatured templateDNA, 50 pmoles of each oligonucleotide primer, 2.5 U of Taq polymerase,and 10% DMSO). The reactions usually contain 150 to 320 μM each of dATP,dCTP, dTTP, dGTP, or one or more analogs thereof.

The newly synthesized strands form a double-stranded molecule that canbe used in the succeeding steps of the reaction. The steps of strandseparation, annealing, and elongation can be repeated as often as neededto produce the desired quantity of amplification products correspondingto the target GBS nucleic acid molecule. The limiting factors in thereaction are the amounts of primers, thermostable enzyme, and nucleosidetriphosphates present in the reaction. The cycling steps (i.e.,denaturation, annealing, and extension) are preferably repeated at leastonce. For use in detection, the number of cycling steps will depend,e.g., on the nature of the sample. If the sample is a complex mixture ofnucleic acids, more cycling steps will be required to amplify the targetsequence sufficient for detection. Generally, the cycling steps arerepeated at least about 20 times, but may be repeated as many as 40, 60,or even 100 times.

Fluorescence Resonance Energy Transfer (FRET)

FRET technology (see, for example, U.S. Pat. Nos. 4,996,143, 5,565,322,5,849,489, and 6,162,603) is based on a concept that when a donor and acorresponding acceptor fluorescent moiety are positioned within acertain distance of each other, energy transfer takes place between thetwo fluorescent moieties that can be visualized or otherwise detectedand/or quantitated. Two oligonucleotide probes, each containing afluorescent moiety, can hybridize to an amplification product atparticular positions determined by the complementarity of theoligonucleotide probes to the GBS target nucleic acid sequence. Uponhybridization of the oligonucleotide probes to the amplification productnucleic acid at the appropriate positions, a FRET signal is generated.Hybridization temperatures can range from about 35 to about 65° C. forabout 10 secs to about 1 min.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range.

As used herein with respect to donor and corresponding acceptorfluorescent moieties “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radiative energytransfer can be produced therebetween.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

Representative donor fluorescent moieties that can be used with variousacceptor fluorescent moieties in FRET technology include fluorescein,Lucifer Yellow, B-phycoerythrin, 9-acridineisothiocyanate, LuciferYellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC™-Red 640, LC™-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

The donor and acceptor fluorescent moieties can be attached to theappropriate probe oligonucleotide via a linker arm. The length of eachlinker arm is important, as the linker arms will affect the distancebetween the donor and acceptor fluorescent moieties. The length of alinker arm for the purpose of the present invention is the distance inAngstroms (Å) from the nucleotide base to the fluorescent moiety. Ingeneral, a linker arm is from about 10 to about 25 Å. The linker arm maybe of the kind described in WO 84/03285. WO 84/03285 also disclosesmethods for attaching linker arms to a particular nucleotide base, andalso for attaching fluorescent moieties to a linker arm.

An acceptor fluorescent moiety such as an LC™-Red 640-NHS-ester can becombined with C6-Phosphoramidites (available from ABI (Foster City,Calif.) or Glen Research (Sterling, Va.)) to produce, for example,LC™-Red 640-Phosphoramidite. Frequently used linkers to couple a donorfluorescent moiety such as fluorescein to an oligonucleotide includethiourea linkers (FITC-derived, for example, fluorescein-CPG's from GlenResearch or ChemGene (Ashland, Mass.)), amide-linkers(fluorescein-NHS-ester-derived, such as fluorescein-CPG from BioGenex(San Ramon, Calif.)), or 3′-amino-CPG's that require coupling of afluorescein-NHS-ester after oligonucleotide synthesis.

Detection of Group B Streptococcus

The presence of GBS has been detected by culturing the organism as wellas rapid antigen tests. Conventional PCR methods also have been used todetect GBS. Conventional PCR-based amplification is generally followedby transfer of the amplification products to a solid support anddetection using a labeled probe (e.g., a Southern or Northern blot).These methods are labor intensive and frequently require more than oneday to complete. Additionally, the manipulation of amplificationproducts for the purpose of detection (e.g., by blotting) increases therisk of carry-over contamination and false positives.

By using commercially available real-time PCR instrumentation (e.g.,LightCycler™, Roche Molecular Biochemicals, Indianapolis, Ind.), PCRamplification and detection of the amplification product can be combinedin a single closed cuvette with dramatically reduced cycling time. Sincedetection occurs concurrently with amplification, the real-time PCRmethods obviate the need for manipulation of the amplification product,and diminish the risk of cross-contamination between amplificationproducts. Real-time PCR greatly reduces turn-around time and is anattractive alternative to conventional PCR techniques in the clinicallaboratory.

The present invention provides methods for detecting the presence orabsence of GBS in a biological sample from an individual or in anon-biological sample. Methods provided by the invention avoid problemsof sample contamination, false negatives, and false positives. Themethods include performing at least one cycling step that includesamplifying a GBS portion of a pts nucleic acid molecule from a sampleusing a pair of pts primers. Each of the pts primers anneals to a targetwithin or adjacent to a GBS pts nucleic acid molecule such that at leasta portion of each amplification product contains nucleic acid sequencecorresponding to pts. More importantly, the amplification product shouldcontain the nucleic acid sequences that are complementary to the ptsprobes. The pts amplification product is produced provided that GBSnucleic acid is present. Each cycling step further includes contactingthe sample with a pair of pts probes. According to the invention, onemember of each pair of the pts probes is labeled with a donorfluorescent moiety and the other is labeled with a correspondingacceptor fluorescent moiety. The presence or absence of FRET between thedonor fluorescent moiety of the first pts probe and the correspondingacceptor fluorescent moiety of the second pts probe is detected uponhybridization of the pts probes to the pts amplification product.

Each cycling step includes an amplification step and a hybridizationstep, and each cycling step is usually followed by a FRET detectingstep. Multiple cycling steps are performed, preferably in athermocycler. Methods of the invention can be performed using the ptsprimer and probe set to detect the presence of GBS. Detection of FRET inthe pts reaction indicates the presence of a GBS.

As used herein, “amplifying” refers to the process of synthesizingnucleic acid molecules that are complementary to one or both strands ofa template nucleic acid molecule (e.g., GBS pts nucleic acid molecules).Amplifying a nucleic acid molecule typically includes denaturing thetemplate nucleic acid, annealing primers to the template nucleic acid ata temperature that is below the melting temperatures of the primers, andenzymatically elongating from the primers to generate an amplificationproduct. Amplification typically requires the presence ofdeoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g.,Platinum® Taq) and an appropriate buffer and/or co-factors for optimalactivity of the polymerase enzyme (e.g., MgCl₂ and/or KCl).

If amplification of GBS nucleic acid occurs and an amplification productis produced, the step of hybridizing results in a detectable signalbased upon FRET between the members of the pair of probes. As usedherein, “hybridizing” refers to the annealing of probes to anamplification product. Hybridization conditions typically include atemperature that is below the melting temperature of the probes but thatavoids non-specific hybridization of the probes.

Generally, the presence of FRET indicates the presence of GBS in thesample, and the absence of FRET indicates the absence of GBS in thesample. Inadequate specimen collection, transportation delays,inappropriate transportation conditions, or use of certain collectionswabs (calcium alginate or aluminum shaft) are all conditions that canaffect the success and/or accuracy of a test result, however. Using themethods disclosed herein, detection of FRET within 45 cycling steps isindicative of a GBS infection.

Methods of the invention also can be used for GBS vaccine efficacystudies or epidemiology studies. For example, an attenuated GBS in ananthrax vaccine can be detected using the methods of the inventionduring the time when bacteria is still present in an individual. Forsuch vaccine efficacy studies, the methods of the invention can be usedto determine, for example, the persistence of an attenuated strain ofGBS used in a vaccine, or can be performed in conjunction with anadditional assay such as a serologic assay to monitor an individual'simmune response to such a vaccine. In addition, methods of the inventioncan be used to distinguish one GBS strain from another for epidemiologystudies of, for example, the origin or severity of an outbreak of GBS.

Representative biological samples that can be used in practicing themethods of the invention include dermal swabs, cerebrospinal fluid,blood, sputum, bronchio-alveolar lavage, bronchial aspirates, lungtissue, and feces. Collection and storage methods of biological samplesare known to those of skill in the art. Biological samples can beprocessed (e.g., by nucleic acid extraction methods and/or kits known inthe art) to release GBS nucleic acid or in some cases, the biologicalsample can be contacted directly with the PCR reaction components andthe appropriate oligonucleotides.

Biological samples can be cultured in a medium suitable for growth ofGBS. The culture media then can be assayed for the presence or absenceof GBS using the methods of the invention as described herein. Forexample, samples arriving at a clinical laboratory for detection of GBSusing the methods of the invention can be in the form of a liquidculture that had been inoculated with a biological sample from anindividual.

Melting curve analysis is an additional step that can be included in acycling profile. Melting curve analysis is based on the fact that DNAmelts at a characteristic temperature called the melting temperature(Tm), which is defined as the temperature at which half of the DNAduplexes have separated into single strands. The melting temperature ofa DNA depends primarily upon its nucleotide composition. Thus, DNAmolecules rich in G and C nucleotides have a higher Tm than those havingan abundance of A and T nucleotides. By detecting the temperature atwhich signal is lost, the melting temperature of probes can bedetermined. Similarly, by detecting the temperature at which signal isgenerated, the annealing temperature of probes can be determined. Themelting temperature(s) of the pts probes from the pts amplificationproduct can confirm the presence or absence of GBS in the sample.

Within each thermocycler run, control samples are cycled as well.Positive control samples can amplify GBS nucleic acid control template(other than pts) using, for example, control primers and control probes.Positive control samples can also amplify, for example, a plasmidconstruct containing GBS pts nucleic acid molecules. Such a plasmidcontrol can be amplified internally (e.g., within the sample) or in aseparate sample run side-by-side with the patients' samples. Eachthermocycler run should also include a negative control that, forexample, lacks GBS template DNA. Such controls are indicators of thesuccess or failure of the amplification, hybridization and/or FRETreaction. Therefore, control reactions can readily determine, forexample, the ability of primers to anneal with sequence-specificity andto initiate elongation, as well as the ability of probes to hybridizewith sequence-specificity and for FRET to occur.

In an embodiment, the methods of the invention include steps to avoidcontamination. For example, an enzymatic method utilizing uracil-DNAglycosylase is described in U.S. Pat. Nos. 5,035,996, 5,683,896 and5,945,313 to reduce or eliminate contamination between one thermocyclerrun and the next. In addition, standard laboratory containment practicesand procedures are desirable when performing methods of the invention.Containment practices and procedures include, but are not limited to,separate work areas for different steps of a method, containment hoods,barrier filter pipette tips and dedicated air displacement pipettes.Consistent containment practices and procedures by personnel arenecessary for accuracy in a diagnostic laboratory handling clinicalsamples.

Conventional PCR methods in conjunction with FRET technology can be usedto practice the methods of the invention. In one embodiment, aLightCycler™ instrument is used. A detailed description of theLightCycler™ System and real-time and on-line monitoring of PCR can befound at biochem.roche.com/lightcycler on the World Wide Web. Thefollowing patent applications describe real-time PCR as used in theLightCycler™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. TheLightCycler™ instrument is a rapid thermal cycler combined with amicrovolume fluorometer utilizing high quality optics. This rapidthermocycling technique uses thin glass cuvettes as reaction vessels.Heating and cooling of the reaction chamber are controlled byalternating heated and ambient air. Due to the low mass of air and thehigh ratio of surface area to volume of the cuvettes, very rapidtemperature exchange rates can be achieved within the LightCycler™thermal chamber. Addition of selected fluorescent dyes to the reactioncomponents allows the PCR to be monitored in real time and on-line.Furthermore, the cuvettes serve as an optical element for signalcollection (similar to glass fiber optics), concentrating the signal atthe tip of the cuvette. The effect is efficient illumination andfluorescent monitoring of microvolume samples.

The LightCycler™ carousel that houses the cuvettes can be removed fromthe instrument. Therefore, samples can be loaded outside of theinstrument (in a PCR Clean Room, for example). In addition, this featureallows for the sample carousel to be easily cleaned and sterilized. Thefluorometer, as part of the LightCycler™ apparatus, houses the lightsource. The emitted light is filtered and focused by an epi-illuminationlens onto the top of the cuvette. Fluorescent light emitted from thesample is then focused by the same lens, passed through a dichroicmirror, filtered appropriately, and focused onto data-collectingphotohybrids. The optical unit currently available in the LightCycler™instrument (Roche Molecular Biochemicals, Catalog No. 2 011 468)includes three band-pass filters (530 nm, 640 nm, and 710 nm), providingthree-color detection and several fluorescence acquisition options. Datacollection options include once per cycling step monitoring, fullycontinuous single-sample acquisition for melting curve analysis,continuous sampling (in which sampling frequency is dependent on samplenumber) and/or stepwise measurement of all samples after definedtemperature interval.

The LightCycler™ can be operated using a PC workstation and can utilizea Windows NT operating system. Signals from the samples are obtained asthe machine positions the capillaries sequentially over the opticalunit. The software can display the fluorescence signals in real-timeimmediately after each measurement. Fluorescent acquisition time is10-100 milliseconds (msec). After each cycling step, a quantitativedisplay of fluorescence vs. cycle number can be continually updated forall samples. The data generated can be stored for further analysis.

As an alternative to FRET, an amplification product can be detectedusing a double-stranded DNA binding dye such as a fluorescent DNAbinding dye (e.g., SYBRGreenI® or SYBRGold® (Molecular Probes)). Uponinteraction with the double-stranded nucleic acid, such fluorescent DNAbinding dyes emit a fluorescence signal after excitation with light at asuitable wavelength. A double-stranded DNA binding dye such as a nucleicacid intercalating dye also can be used. When double-stranded DNAbinding dyes are used, a melting curve analysis is usually performed forconfirmation of the presence of the amplification product.

As described herein, amplification products also can be detected usinglabeled hybridization probes that take advantage of FRET technology. Acommon format of FRET technology utilizes two hybridization probes. Eachprobe can be labeled with a different fluorescent moiety and aregenerally designed to hybridize in close proximity to each other in atarget DNA molecule (e.g., an amplification product). A donorfluorescent moiety, for example, fluorescein, is excited at 470 nm bythe light source of the LightCycler™ Instrument. During FRET, thefluorescein transfers its energy to an acceptor fluorescent moiety suchas LightCycler™-Red 640 (LC™-Red 640) or LightCycler™-Red 705 (LC™-Red705). The acceptor fluorescent moiety then emits light of a longerwavelength, which is detected by the optical detection system of theLightCycler™ instrument. Efficient FRET can only take place when thefluorescent moieties are in direct local proximity and when the emissionspectrum of the donor fluorescent moiety overlaps with the absorptionspectrum of the acceptor fluorescent moiety. The intensity of theemitted signal can be correlated with the number of original target DNAmolecules (e.g., the number of GBS genomes).

Another FRET format utilizes TaqMan® technology to detect the presenceor absence of an amplification product, and hence, the presence orabsence of GBS. TaqMan® technology utilizes one single-strandedhybridization probe labeled with two fluorescent moieties. When a firstfluorescent moiety is excited with light of a suitable wavelength, theabsorbed energy is transferred to a second fluorescent moiety accordingto the principles of FRET. The second fluorescent moiety is generally aquencher molecule. During the annealing step of the PCR reaction, thelabeled hybridization probe binds to the target DNA (i.e., theamplification product) and is degraded by the 5′ to 3′ exonucleaseactivity of the Taq Polymerase during the subsequent elongation phase.As a result, the excited fluorescent moiety and the quencher moietybecome spatially separated from one another. As a consequence, uponexcitation of the first fluorescent moiety in the absence of thequencher, the fluorescence emission from the first fluorescent moietycan be detected. By way of example, an ABI PRISM® 7700 SequenceDetection System (Applied Biosystems, Foster City, Calif.) uses TaqMan®technology, and is suitable for performing the methods described hereinfor detecting GBS. Information on PCR amplification and detection usingan ABI PRISM® 770 system can be found at appliedbiosystems.com/productson the World Wide Web.

Molecular beacons in conjunction with FRET also can be used to detectthe presence of an amplification product using the real-time PCR methodsof the invention. Molecular beacon technology uses a hybridization probelabeled with a first fluorescent moiety and a second fluorescent moiety.The second fluorescent moiety is generally a quencher, and thefluorescent labels are typically located at each end of the probe.Molecular beacon technology uses a probe oligonucleotide havingsequences that permit secondary structure formation (e.g., a hairpin).As a result of secondary structure formation within the probe, bothfluorescent moieties are in spatial proximity when the probe is insolution. After hybridization to the target nucleic acids (i.e.,amplification products), the secondary structure of the probe isdisrupted and the fluorescent moieties become separated from one anothersuch that after excitation with light of a suitable wavelength, theemission of the first fluorescent moiety can be detected.

It is understood that the present invention is not limited by theconfiguration of one or more commercially available instruments.

Articles of Manufacture

The invention further provides for articles of manufacture to detectGBS. An article of manufacture according to the present invention caninclude primers and probes used to detect GBS, together with suitablepackaging materials. Representative primers and probes for detection ofGBS are capable of hybridizing to GBS pts nucleic acid molecules.Methods of designing primers and probes are disclosed herein, andrepresentative examples of primers and probes that amplify and hybridizeto GBS pts nucleic acid molecules are provided.

Articles of manufacture of the invention also can include one or morefluorescent moieties for labeling the probes or, alternatively, theprobes supplied with the kit can be labeled. For example, an article ofmanufacture may include a donor fluorescent moiety for labeling one ofthe pts probes and an acceptor fluorescent moiety for labeling the otherpts probe, respectively. Examples of suitable FRET donor fluorescentmoieties and corresponding acceptor fluorescent moieties are providedabove.

Articles of manufacture of the invention also can contain a packageinsert or package label having instructions thereon for using the ptsprimers and probes to detect GBS in a sample. Articles of manufacturemay additionally include reagents for carrying out the methods disclosedherein (e.g., buffers, polymerase enzymes, co-factors, or agents toprevent contamination). Such reagents may be specific for one of thecommercially available instruments described herein.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 LightCycler Detection of Group B Streptococcus

A LightCycler assay was used to detect group B streptococcus (GBS)bacterial pathogens from vaginal/anal swabs. A conserved region of thephosphotransferase gene (pts) of GBS was used as a target for the PCRassay detection.

The primer sequences were as follows: primer 1: 5′-TGA GAA GGC AGT AGAAAG CTT AG-3′ (SEQ ID NO:1); and primer 2: 5′-TGC ATG TAT GGG TTA TCTTCC-3′ (SEQ ID NO:2). The probe sequences and labels were as follows:probe 1: 5′-CAA ATT AAA GAG ACT ATT CGT GCA A-fluorescein-3′ (SEQ IDNO:3); and probe 2: 5′-LC-Red640-CAA GTA AAT GCA GAA ACA GG-phosphate-3′(SEQ ID NO:4).

Primers were adjusted to 50 μM by measuring the A₂₆₀ of a 1/50 dilution(196 μl water+4 μl, Dilution Factor (DF)=50). The concentration wasestimated by the following formula:(μM found/50)×μl remaining)−μl remaining=water to add

Probes were dissolved in TE′ to a concentration of 20 μM (supplied withthe probes and resuspended according to manufacturer's instructions).The concentration of oligonucleotides and dye was double checked by UVabsorption using the following equations from Biochemica, 1999, 1:5-8:

$\lbrack{dye}\rbrack = {{\frac{A_{dye}}{E_{dye}}\mspace{14mu}\lbrack{oligo}\rbrack} = \frac{A_{260} - \left( {A_{260} \times \frac{E_{260{({dye})}}}{E_{dye}}} \right)}{\frac{10^{6}}{{nmol}/A_{260}}}}$Absorbance Emission Abs max E_(dye) E_(260(dye)) Max Dye (nm) (M⁻¹cm⁻¹)(M⁻¹cm⁻¹) (nm) Fluorescein 494 68,000 2,000 524 LC Red 640 622 110,00031,000 638

Table 1 shows the PCR Reaction Mix.

TABLE 1 Ingredient Stock Concentration μl Water 11 MgCl₂ 50 mM 1.2LC-FS-DNA MHP* 10X 2 Primer 1 25 mM 0.24 Primer 2 25 mM 0.24 FluoresceinProbe 20 μM 0.2 Red640 Probe 20 μM 0.2 Total Volume 15 *LC-FS-DNA MHP =LightCycler FastStart DNA Master Hybridization Probes (Roche, CatalogNo. 3003248)

5 μl of the swab sample was mixed with 15 μl of the PCR Reaction Mix andadded to the LightCycler cuvette for thermocycling. The samples were PCRamplified and the products were detected in a LightCycler instrument(Roche Applied Science, catalog 2011468). The PCR cycling procedure usedis shown in Table 2.

TABLE 2 PCR Hold Time Temperature Slope Signal program Cycles (seconds)(° C.) (° C./sec) acquisition Initial 1 600 95 20 None PCR 45 10 95 20None 15 55 20 Single 15 72 20 None Melting 1 0 95 20 None curve 20 59 20None analysis 20 45 0.2 None 0 85 0.2 Continuous Cool 1 10 40 20 None

The signal detected in the 640 nm channel of the LightCycler instrumentwas analyzed. A melting curve analysis, performed after the PCRamplification, was used to confirm detection of GBS. GBS positivesamples demonstrate a Tm of 58° C. plus or minus 2° C., and werecompared directly to the positive control. Negative samples had nomelting curve.

Plasmid controls were produced by cloning the pts product amplified bythe pts primers into a plasmid (TA Cloning® Kit, Invitrogen, Carlsbad,Calif.). The plasmid containing the target insert was used to determinethe analytical sensitivity of the assays. Plasmid concentration or thecopy number of the gene target insert was determined with the followingformula:DS DNA, A₂₆₀ to molecules/μl

Given:(A ₂₆₀×Dilution Factor)/20=mg/ml=μg/μl DS DNA  1.

-   -   1 A₂₆₀=50 μg/ml    -   1 A₂₆₀ (50)=μg/ml    -   1 A₂₆₀ (50)/1000=μg/μl        (6.02×10²³ molecules/mole)/(10¹² pmole/mole)=6.02×10¹¹        molecules/pmole  2.        Base pairs of DNA in molecule=N  3.

Then:(A ₂₆₀×DF)/20 μg/μl×10⁶ pg/μg×1 pmol/660 pg×1/N×6.02×10¹¹molecules/pmole=molecules/μl

Shortcut Calculation:((A ₂₆₀ ×DF)/20)×(9.12×10¹⁴ /N)=molecules/μl

Example 2 Specificity of LightCycler Assay of GBS

DNA extracted from cultures of a variety of different organisms wereused to determine if the GBS assay would cross-react with non-GBSorganisms. Organisms similar to GBS were tested as well as organismscommonly found in a vaginal or anal swab sample were tested. Table 3shows the similar organisms tested and the results. Table 4 shows theother specimens tested and those results.

TABLE 3 Lancefield Source Organism Group ATCC Other LC assay S. pyogenesA 19615 Neg S. agalactiae B CAPXL36 Pos S. suis 43765 Neg L. lactis19435 Neg S. equi ss equi C 33398 Neg 51° C. melt S. uberis 19436 Neg S.canis G 43496 Neg 51° C. melt E. faecium CAP-D-18-83 Neg S. bovisCAP-D-16-83 Neg E. faecalis 29212 Neg S. dysgalactiae C 43078 Neg 51° C.melt S. salivarius 7073 Neg S. equinus 9812 Neg S. pneumoniae 49619 NegS. porciuns 43138 Neg S. iniae 29178 Neg S. anginosus 33397 Neg S.MG-intermedius CAP-D-17-87 Neg Group F strep F SCB-21-89 Neg S. sanguisSCB-33-83 Neg S. mitis 49456 Neg S. oralis 35037 Neg S. gordonii 10558Neg S. mutans QC strain - Neg Mayo S. intermdius 27335 Neg S. anginosus33397 Neg

TABLE 4 Respiratory Organism Source LC result Acinetobacter bauminiipatient isolate Neg Acinetobacter lwoffii QC Strain Neg Aeromonashydrophilia CAP-D-1-82 Neg Bordetella bronchioseptica patient isolateNeg Bordetella parapertussis ATCC 15311 Neg Campylobacter jejuniCDC-AB2-C15- Neg 82 Corynebacterium patient isolate Neg(Archanobacterium) haemolyticum Corynebacterium diptheriae SCB-25-86 NegCorynebacterium NY-4-88 Neg pseudodiptheriae Escherichia coli patientisolate Neg Haemophilus influenza ATCC 49766 Neg Human DNA MRC-5 cellsNeg Klebsiella oxytoca patient isolate Neg Klebsiella pneumoniae patientisolate Neg Legionella jordanis ATCC 33623 Neg Legionella pneumophilaATCC 33152 Neg Listeria monocytogenes patient isolate Neg Moraxellacatarrhalis patient isolate Neg Morganella morganii CAP-D-5-79 NegMycoplasma pneumoniae patient isolate Neg Neiserria gonorrheae patientisolate Neg Neiserria meningitides patient isolate Neg Proteus vulgarispatient isolate Neg Pseudomonas cepacia patient isolate Neg Pseudomonasfluorescens patient isolate Neg Staphylococcus aureus ATCC 25923 NegStaphylococcus epidermidis patient isolate Neg Stenotrophomonasmaltophilia SOB-33-77 Neg Citrobacter freundii patient isolate NegBordetella bronchioseptica ATCC 19395 Neg Stool panel LC Organism sourceassay Actinomyces pyogenes clinical Neg Aeromonas hydrophila CAP-D-1-82Neg Bacteroides distasonis ATCC 8503 Neg Bacteroides fragilis ATCC 25285Neg Bacteroides thetaiotaomicron ATCC 29741 Neg Bacteroides vulgatusATCC 29327 Neg Citrobacter freundii clinical Neg Clostridium perfingensATCC 13124, Neg Cl417 E. coli O70:K:H42 ATCC 23533 Neg Enterobactercloacae clinical-1004 Neg Enterococcus faecalis clinical V583 NegEnterococcus faecium clinical B7641 Neg Escherichia hermanii clinicalNeg Escherichia vulneris clinical Neg Eubacterium lentum ATCC 43055 NegFusobacterium nucleatum ATCC 25559 Neg Klebsiella pneumoniae ATCC 700603Neg Proteus mirabilis QC strain Neg Pseudomonas aeruginosa ATCC 27853Neg Genital-Urinary panel LC Organism Source Result Acinetobacter lwofficlinical Neg Candida albicans clinical Neg Neisseria gonorrheae clinicalNeg Corynebacterium ATCC 10700 Neg pseudotubercuolsis Gardnerellavaginalis clinical Neg Mobiluncus curtissi clinical Neg Mycoplasmaclinical Neg Neisseria lactamica clinical Neg Peptostreptococcus magnusclinical Neg Porphyromonas gingivalis clinical Neg Prevotella biviaclinical Neg Ureoplasma clinical Neg

Example 3 Analytical Sensitivity of the LightCycler Assay

As few as 5 copies of GBS target DNA per reaction were detected by theGBS LightCycler assay.

Example 4 Clinical Sensitivity of the LightCycler Assay

Prior to the LightCycler technology, the gold standard for detection ofGBS was culture. Culture results from vaginal/anal swab specimens fromwomen collected during the 35 to 37 week of pregnancy were compared tothe LightCycler GBS assay.

Culture LC Present Absent Totals Positive 37 4 41 Negative 0 134 134Totals 37 138 175

The results below were calculated using StatsDirect version 1.9.15software (StatsDirect Ltd, Cheshire, UK) and include 95% confidenceintervals (shown in parentheses). An explanation of the values shownbelow and how those values are calculated can be found atmusc.edu/dc/icrebm/sensitivity.html on the World Wide Web.

Disease Present Absent Test + a (true) b (false) − c (false) d (true)Prevalence (percent of affected patients tested; [a+c/d]):

21.14% (15.34% to 27.95%)

Positive predictive value (percent of patients with a positive testhaving the disease; [a/a+b]):

90.24% (76.87% to 97.28%)

Negative predictive value (percent of patients with a negative testwithout the disease; [d/d+c]):

100% (97.28% to *%)

Sensitivity (true positives detected per total affected patients tested;[a/a+c]):

100% (90.51% to *%)

Specificity (true negatives per unaffected patients tested; [d/b+d]):

97.1% (92.74% to 99.2%)

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. An article of manufacture, comprising a pair of pts primers, a pairof pts probes, a donor fluorescent moiety and a corresponding acceptorfluorescent moiety, wherein said pair of pts primers comprises a firstpts primer and a second pts primer, wherein said first pts primerconsists of the sequence 5′-TGA GAA GGC AGT AGA AAG CTT AG-3′ (SEQ IDNO:1), and wherein said second pts primer consists of the sequence5′-TGC ATG TAT GGG TTA TCT TCC-3′ (SEQ ID NO:2), wherein said pair ofpts probes comprises a first pts probe and a second pts probe, whereinsaid first pts probe consists of the sequence 5′-CAA ATT AAA GAG ACT ATTCGT GCA A-3′ (SEQ ID NO:3), and wherein said second pts probe consistsof the sequence 5′-CAA GTA AAT GCA GAA ACA GG-3′ (SEQ ID NO:4).
 2. Thearticle of manufacture of claim 1, wherein said first pts probe islabeled with said donor fluorescent moiety and wherein said second ptsprobe is labeled with said corresponding acceptor fluorescent moiety. 3.The article of manufacture of claim 2, further comprising a packageinsert having instructions thereon for using said pair of pts primersand said pair of pts probes to detect the presence or absence of Group BStreptococcus (GBS) in a sample.